Our objective is to investigate the role of protein translation in matrix accumulation, a major contributor to loss of renal function in type 2 diabetes. The rationale is based on our observation that the insulin receptor (IR) and its specific signal transduction pathways are activated in the renal cortex of db/db mice in early stages of type 2 diabetes at a time when the mice are hyperinsulinemic. At this time, increased matrix protein accumulation is associated with decrease in matrix protein laminin mRNA. Furthermore, in renal cells in vitro, insulin increases matrix laminin synthesis by augmenting its translation and activating the same signaling pathways as seen in the renal cortex of db/db mice. Insulin activates eukaryotic initiation factor 4E (elF4E) and elongation factor, eEF2 that critically regulate protein translation. Central hypothesis: In type 2 diabetes, hyperinsulinemia stimulates specific signaling pathways leading to activation of factors controlling protein translation, resulting in accumulation of matrix in the kidney. We will study db/db mice in pre-diabetic, early, and established phases of diabetes, with appropriate controls. Specific Aim I: In vivo studies To identify IR signal transduction pathways involved in activation of elF4E, eEF2 and augmented matrix translation in renal cortex of mice with type 2 diabetes. We will assess phosphorylation of elF4E, its binding protein 4E-BP1, and eEF2. Polyribosomal analysis will directly examine translation initiation of matrix protein laminin. Activation of IR and insulin-stimulated kinases that regulate protein translation, i.e., PI 3-kinase, Akt, p70s6k, Erk-1/-2 type MAP kinase and PKC will be studied to establish the role of these signals in activating translation. Specific Aim II: In vitro studies To identify IR signaling kinases required for augmenting matrix translation employing proximal tubular epithelial (PTE) and mesangial cells. Role of individual kinases will be assessed by employing specific inhibitors, antisense oligos and dominant negative mutants in renal cells, in a milieu simulating type 2 diabetes. Specific Aim III: In vivo studies To evaluate the role of PTE cell-IR in activation of signaling pathways leading to augmented matrix translation in renal cortex of mice with type 2 diabetes by targeted IR gene deletion. PTE cell-specific IR deletion will be accomplished using Cre/LoxP system. We will generate a transgenic mouse on db/db background, in which Cre recombinase is driven by a PTE-cell specific kidney androgen regulated protein (KAP) promoter. IR (lox/+) mice will be obtained from Dr. C. Ronald Kahn. Hybrids of the IR (lox/+) and KAP-Cre mice will be used to generate kidney IR knockout mice. Functional (GFR, proteinuria), histologic (matrix proteins), IR signaling and protein translation events in these mice will be compared with those in wild type db/db mice. This strategy permits a definitive assessment of role of PTE celMR in pathogenesis of nephropathy in type 2 diabetes. Novel mechanistic data on renal disease in type 2 diabetes is expected, and may lead to unique therapeutic strategies.